The mTORC1 pathway regulates multiple cellular processes to promote a switch from catabolic to anabolic metabolism and is thus under tight regulatory control by growth factor signaling and nutrient sensing pathways. Dysregulation of this complex machinery is implicated in many cancers, so defining the key mechanisms by which mTORC1 senses changes in cellular homeostasis to activate growth signals is of great relevance. Spatial regulation of mTORC1 signaling has been recognized as a major mechanism that influences the cellular response to nutrients and the lysosome is central to this process. We found that the tumor suppressor TMEM127, a poorly characterized lysosomal protein, is a component of the lysosome-anchored multiprotein complex involved in the mTORC1 response to amino acids. In this proposal we seek to define the mechanisms through which the interaction between TMEM127 and the mTORC1 regulatory machinery is regulated and how this can impact on mTORC1 inhibition. We previously identified TMEM127 as a tumor suppressor gene mutated in hereditary neuroendocrine tumors, pheochromocytomas, and in renal cancers, and found that mutant tumors display increased mTORC1 signaling. Our earlier studies revealed that TMEM127 loss leads to lysosomal expansion with redistribution of mTOR toward the lysosome. The lysosome functions as a docking platform for mTORC1 signaling in response to amino acids through the assembly of a multi?protein complex involving Rag GTPases, Ragulator (LAMTOR1?5) and vacuolar ATPase (v?ATPase). Using multiple in vivo and in vitro approaches developed in our lab, we found that TMEM127 physically associates with ragulator, vATPase and Rags, and in its absence the interaction between ragulator and Rags is enhanced and mTOR recruitment by Rags is increased. Furthermore, our preliminary data support of an effect of TMEM127 in nutrient sensing. Based on these observations, our general hypothesis is that TMEM127 disrupts mTOR recruitment to the lysosomal?centered protein complex through inhibition of the ragulator-Rag interaction in response to amino acids. To test this hypothesis, we propose to define the signals that regulate the TMEM127-ragulator-Rag-mTORC1 interaction, including its response to individual amino acids, to other nutrients (e.g. glucose), to cellular stresses including starvation, osmolality, oxidative stress, and growth factor signaling. Furthermore, we propose to systematically define the components of the lysosomal multiprotein assembly that are required for TMEM127 binding, and conversely, which domains of TMEM127 are necessary for this interaction. Finally, we will explore the emerging notion that the nutrient sensing and the growth-factor signaling branches of mTORC1 activation are integrated at the lysosome by defining TMEM127's contribution to the mTOR inhibitory actions by TSC2 at the lysosomal surface.To carry out these experiments we will take advantage of tools and models that were developed in our lab, i.e. an in vivo mouse strain of targeted Tmem127 deletion, cell models of human TMEM127 knockout (by CRISPR-Cas9-based genome editing), knockdown (by siRNA), as well as a cohort of primary tumor samples with TMEM127 mutations, along with mutant constructs and genome edited cells that mimic naturally-occurring TMEM127 mutations identified in patients with cancer. In addition, genetic models of activation or loss of ragulator, Rags and mTOR components will complement our analysis. Given its tumor suppressor role in humans, understanding the contribution of TMEM127, a novel lysosomal protein that participates in the mTORC1 lysosomal assembly should provide insights into how nutrient and growth signals are integrated and can be coopted by cancer cells to promote uncontrolled proliferation.